2.4 Preparation of
Samples for LC-MS
1. Fresh Milli-Q water (see Note 4) + 0.2% formic acid.
2. LC-MS grade acetonitrile + 0.2% formic acid.
3. Vortexer.
4. Temperature-controlled microcentrifuge.
5. LC-MS sampling vials with insert and rubber-seal snap caps.
6. LC-MS autosampler tray (see Note 12).
2.5 Standard
Reversed-Phase LCMS Run
1. Water–formic acid (see Subheading 2.4, item 1).
2. Acetonitrile–formic acid (see Subheading 2.4, item 2).
3. LC-MS system, such as a Cogent Diamond Hydride Type C
column linked to an Agilent Accurate Mass 6220 Time-ofFlight (TOF) spectrometer and coupled with an Agilent 1200
LC system (or equivalent) (see Note 12).
4. Reference mass solution, as appropriate.
2.6 Ion-Pairing
Reversed-Phase LCMS Run
1. 97:3 Milli-Q water:LC-MS grade methanol (see Note 4) containing 5 mM tributylamine (TBA) and 5.5 mM acetic acid.
2. LC-MS grade methanol containing 5 mM (TBA) and 5.5 mM
acetic acid.
3. LC-MS system, such as an Agilent Zorbax Extend C18 column
linked to an Agilent Accurate Mass 6220 Time-of-Flight
(TOF) spectrometer and coupled with an Agilent 1200 LC
system (or equivalent) (see Note 12).
4. Reference mass solution, as appropriate.
2.7 Activity-Based
Metabolomic Profiling
(ABMP) Sample
Preparation (See Note
15)
1. 7H9-ADN medium (see Subheading 2.1, item 1).
2. Centrifuge.
3. Centrifuge buckets.
4. Cold metabolite extraction buffer (see Subheading 2.2, item
2).
5. Purified enzyme of interest (in a dilute, chemically stable
buffer).
6. 20 mM Tris-HCl buffer (pH 7.2).
7. Probe sonicator/ultrasonic homogenizer.
3 Methods
3.1 Culture System
1. In a BSL-3 laboratory, grow or concentrate M. tuberculosis
cultures to a final density of 5 Â 10
8 cells/mL (OD 600 ¼ 1)
in 7H9-ADN medium (see Note 5).
2. Assemble filtration apparatus: Attach the filter holder to the
vacuum flask using a pipette as illustrated (Fig. 3).
584
Kyle A. Planck and Kyu Rhee
Samples for LC-MS
1. Fresh Milli-Q water (see Note 4) + 0.2% formic acid.
2. LC-MS grade acetonitrile + 0.2% formic acid.
3. Vortexer.
4. Temperature-controlled microcentrifuge.
5. LC-MS sampling vials with insert and rubber-seal snap caps.
6. LC-MS autosampler tray (see Note 12).
2.5 Standard
Reversed-Phase LCMS Run
1. Water–formic acid (see Subheading 2.4, item 1).
2. Acetonitrile–formic acid (see Subheading 2.4, item 2).
3. LC-MS system, such as a Cogent Diamond Hydride Type C
column linked to an Agilent Accurate Mass 6220 Time-ofFlight (TOF) spectrometer and coupled with an Agilent 1200
LC system (or equivalent) (see Note 12).
4. Reference mass solution, as appropriate.
2.6 Ion-Pairing
Reversed-Phase LCMS Run
1. 97:3 Milli-Q water:LC-MS grade methanol (see Note 4) containing 5 mM tributylamine (TBA) and 5.5 mM acetic acid.
2. LC-MS grade methanol containing 5 mM (TBA) and 5.5 mM
acetic acid.
3. LC-MS system, such as an Agilent Zorbax Extend C18 column
linked to an Agilent Accurate Mass 6220 Time-of-Flight
(TOF) spectrometer and coupled with an Agilent 1200 LC
system (or equivalent) (see Note 12).
4. Reference mass solution, as appropriate.
2.7 Activity-Based
Metabolomic Profiling
(ABMP) Sample
Preparation (See Note
15)
1. 7H9-ADN medium (see Subheading 2.1, item 1).
2. Centrifuge.
3. Centrifuge buckets.
4. Cold metabolite extraction buffer (see Subheading 2.2, item
2).
5. Purified enzyme of interest (in a dilute, chemically stable
buffer).
6. 20 mM Tris-HCl buffer (pH 7.2).
7. Probe sonicator/ultrasonic homogenizer.
3 Methods
3.1 Culture System
1. In a BSL-3 laboratory, grow or concentrate M. tuberculosis
cultures to a final density of 5 Â 10
8 cells/mL (OD 600 ¼ 1)
in 7H9-ADN medium (see Note 5).
2. Assemble filtration apparatus: Attach the filter holder to the
vacuum flask using a pipette as illustrated (Fig. 3).
584
Kyle A. Planck and Kyu Rhee